Surface proteins of leptospira

ABSTRACT

The invention relates to  Leptospiral  surface proteins, and the nucleic acid molecules which encode them. Various uses are described, including immunoprophylactic, diagnostic and therapeutic methods.

RELATED APPLICATION

This application claims priority of application Ser. No. 60/360,566,filed Feb. 28, 2002, incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to membrane associated proteins of leptospirabacteria, Leptospira Copenhageni in particular. The proteins are usefulboth therapeutically, as, e.g., antisera, immunoprophylactically, asvaccines, as well as diagnostically. They can be used, for example, todetect antibodies in samples taken from subjects suspected of beinginfected, and also to generate antibodies which can then be used todetect the proteins, epitopic portions of the proteins, as well as thebacteria per se. Also a feature of the invention are nucleic acidmolecules encoding these proteins, methods for purifying them, as wellas various applications thereof.

BACKGROUND AND PRIOR ART

leptospira is a genus of bacteria which is a member of the Spirochetesfamily. Other members of this family are Borrelia and Treponema. Allthree genuses are characterized by mobility, and a helical shape. Allmembers of the family cause disease in animals, including humans,livestock, domesticated animals, and wild animals. For example, Borreliais the causative agent for Lyme disease. Other diseases caused bySpirochetes include relapsing fever, syphilis and yaws.

leptospira genus consists of a genetically diverse group of 12 species,eight of which are pathogenic, and four of which are non-pathogenic, andsaprophytic. See Faine, et al., leptospira and Leptospirosis (2^(nd)edition, 1999, Melbourne, Australia, MediSci); Farr, Clin. Infect. Dis21:1-8 (1995). Levett, Clin. Microbiol Rev. 14:296-326 (2001),incorporated by reference. There are over 200 known pathogenic serovarsof Leptospira. It is hypothesized that structural heterogeneity inlypopolysacchacide structure accounts for this. See, Levett, supra.

Leptospirosis, the disease caused by Leptospira, is zoonotic.Transmission to humans results from contact with domestic or wild animalreservoirs, or via contact with animal urine. Infected individuals showa wide spectrum of clinical manifestations in the early phases of theillness including fever, headache, chills and severe myalgia. In 5-15%of the clinical infections, severe multisystem complications result,including jaundice, renal failure and hemorrhaging. See Farr, supra,Faine, et al., supra. Severe leptospirosis has a mortality rate of5-40%.

There is a large spectrum of animal species which serve as reservoirsfor the bacteria. As a result, human leptospirosis is found throughoutthe world, and is considered to be the most widespread zoonotic disease.High risk groups include military personnel, farmers, miners, sewage andwaste removal workers, veterinarians and abattoir workers. See, in thisregard, Levett, supra, Faine, et al., supra. New patterns oftransmission of the disease have emerged; however, emphasizing thepublic health issues associated with this disease. In “developed”countries outbreaks have been associated with recreational diseases,such as white water rafters in Costa Rica, and sporting events involvingextensive outdoor activity, such as triathlons and “Eco-challenges.”See, e.g., Levett, supra; Jackson, Pediatr. Infect. Dis. J 12:48-54(1993); Center For Disease Control and Prevention: Outbreak ofleptospirosis among white water rafters in Costa Rica (1997); Update:Leptospirosis and Unexplained Acute febrile illness among athletesparticipating in triathlons: Ill. and Wis. (1998). Update: Outbreak ofAcute Illness Among Athletes Participating in Eco-Challenge—Sahah2000—Borneo, Malaysia (2000). Underlying conditions associated withpoverty have led to large, urban, epidemics of leptospirosis in Braziland other countries, resulting in high mortality rates. See Lomor, etal., Infect. Dis. Clin. North Am 14:23-39 (2000); Ko et al., Lancet354:820-5 (1999).

Leptospirosis is a major issue in agriculture as well, due to itsassociation with livestock and domestic animals. Among themanifestations of animal leptospirosis are spontaneous abortions, stillbirths, infertility, failure to thrive, reduced milk production, andhigh fatality rates in diverse species such as cows, pigs, sheep, goats,horses and dogs. See Faine, et al., supra. Other manifestations of thedisease are chronic infection, and shedding of pathogenic leptospires.Standard approaches to controlling this include international andnational quarantines in the animal husbandry industry, with negativeeconomic ramifications.

Clearly, there is a need to control leptospirosis; however, efforts havebeen hindered due to a lack of effective approaches. Long term survivalof pathogenic leptospires in soil and water, as well as the abundance ofanimal reservoirs support long term survival of the pathogen soeradication is not a viable option. Hence, efforts have turned toapproaches based upon vaccines.

Currently, available vaccines are based upon inactivated whole cell, ormembrane preparations of pathogenic bacteria. These appear to induceprotective responses via antibody induction. See Levett, supra, Faine,et al, supra. These vaccines do not produce long term protection againstinfection, and they do not confer cross protective immunity againstserovars not included in the vaccines used. The number of serovarspossible, and the cost of multicomponent serovar vaccines have thwarteddevelopment in this area.

A key mechanism in the pathogenesis of leptospirosis, as in otherspirochetal diseases (such as Lyme disease and syphilis), is the abilityof the pathogen to disseminate widely in the host during the early stageof infection. See Faine, et al., supra. It is presumed that surfaceassociated leptospiral proteins, mediate interactions which facilitateentry and dissemination through host tissues. Virulence factorsassociated with the surface of the bacteria serve as vaccine candidates,in that any immune or other protective response would blockdissemination in the host. Another important aspect of surfaceassociated proteins is that they are, literally, “surface-associated,”rendering them accessible to immune attack. Protective mechanismsassociated with such surface associated proteins includeantibody-dependent phagacytosis, and complement-mediated killing.

It would be desirable to have pure, or substantially pure leptospirasurface associated proteins available. Production of such proteins, inrecombinant form, for example is cost effective, and provides a methodto screen proteins to determine sub-unit or epitopic fragment basedvaccines. Further, availability of recombinant proteins permits one todetermine which proteins are conserved, especially among differentpathogenic Leptospira, to determine which are the most suitable,cross-serovar vaccines.

There have been difficulties in identifying surface associatedleptospira proteins using conventional biochemical and molecularbiological methods. The genome of the spirochete Borrelia burgdorferihas been analyzed, and more than 100 surface associated lepoproteinswere identified. The large size of the leptospira genome (−4.6 Mb), andits complex life cycle suggest that a far greater number of surfaceassociated proteins will be found. Using standard membrane extraction,isolation, and purification techniques, less than 10 leptospira surfaceassociated proteins have been identified and characterized. See Haake etal., Infect. Immun 66:1579-1587 (1998); Haake. etal., Infect. Immun.6572-82 (1999); Haake, et al., Infect. Immun 68:2276-2285 (2000); Shanget al., Infect. Immun 63:3174-3181 (1995); Shang. et al., Infect. Immun64:2232-30 (1996). Also see U.S. Pat. Nos. 5,091,301; 5,643,754;5,638,757; 5,824,321; 6,140,083; 6,262,235; 6,306,623; and 6,308,641.All of these articles and patents are incorporated by reference. WhileHaake, et al., Infect. Immun 67:6572-82 (1999), describe immunizationwith recombinant protein L1pL32, OmpLI and LlpL41 but, the response wasnot complete. None of these reference identify virulence associatedproteins.

As has been pointed out, supra, the leptospira genome is large. As aresult, technical difficulties have prevented meaningful results inidentifying surface associated proteins; however, the emerging field ofbioinformatics has placed extremely powerful and valuable tools in thehands of those involved in Leptospiral research.

Via application of the techniques described supra, the inventors havediscovered further leptospira surface associated proteins useful invaccine production, as well as in production of diagnostic kits for usein determining presence, onset, or decrease in Leptospiral infection.These, and other aspects of the invention will be clear from thedisclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-22 inclusive, present data obtained from Western Blottingexperiments, using the recombinant proteins of the invention. In allfigures, control rat serum, and serum from rats that had been immunizedwith Leptospira, and control human sera as well as sera from patientsdiagnosed as suffering from Leptospirosis were used. FIG. 1 shows workdone with the protein of SEQ ID NO: 2 & 4, FIG. 2 with SEQ ID NO: 6 & 8,and so forth.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

A strain of Leptospira, referred to as “Fiocruz L1-30,” was used. Thestrain was isolated from a patient with severe leptospirosis, contractedduring an epidemic in 1996. See Ko. et al., Lancet 354:820-5 (1999),incorporated by reference. leptospires were detected during dark fieldmicroscopy examination of a culture of tween-albumin media that had beeninoculated with patient blood, following “Guidelines for the Control ofLeptospirosis.” WHO Offset Publ. (1982) incorporated by reference. Thestrain was identified as leptospira interrogans, serovar Copenhageni,via biochemical and sera typing analysis. See Ko et al., supra,Barocchi. et al., J. Clin. Microbiol 39:191-195 (2001). A culture of theorganism was then prepared in media with 10% glycerol, and stored at−70° C. Virulence capacity was determined by inoculating 28 day oldweaning hamsters. Kidneys were removed from anesthetized animals,approximately 7 days after infection, macerated and were used toinoculate Tween-albumin media. Any cultures for which growth wasdetected were used to infect additional hamsters, as part of a secondpassage step. In all, three passage steps were performed. Isolates fromthe final passage were used to prepare large scale, two liter cultures,again in Tween albumin media. Following centrifugation (10,000xg for 20minutes), pellets were stored at −70° C., prior to use in DNApurification steps. The genomic DNA was extracted using standardmethods, as set forth in, e.g., Sambrook and Maniatis, MolecularCloning: A Laboratorv Manual (2^(nd) edition, Cold Spring Harbor, 1989).

Once the genomic DNA was isolated, it was partially digested with MboI,in order to generate fragments which ranged from 30 to 60 kilobasepairs. These were cloned into a cosmid vector “Lawrist 4,” described byHanke, et al., Biotechniques 21(4):686-8 and 690-3 (1996), incorporatedby reference to produce a cosmid library. Any dephosphorylated DNAfragments were ligated to the cosmid vector, in the presence of T4 DNAligase. The resulting, recombinant DNA was then packed into phageparticles, using a commercially available product, and was thentransfected into E. coli DH5*. Transfected cells were plated onto solid,Luria-Bertani medium supplemented with kanamycin, and were grown,overnight, at 37° C.

Following culture, the transfectants were used in a shotgun libraryconstruction protocol. To elaborate, genomic DNA was purified, inaccordance with Fleischmann et al., Science 269:496-512 (1995),incorporated by reference, and the purified DNA was sheared intofragments via nebulization.

The resulting fragments were repaired by end filling, using the Klenowfragment of E. coli DNA polymerase, or phage T7 DNA polymerase, or both,before ligation into the SmaI BAP site of pUC 18. The resulting,recombinant DNA was transformed into E. coli DH 10B cells, viaelectroporation. The transformants were plated, on Luria-Bertani agarmedium containing ampicillin, and grown overnight at 37° C.

Single bacterial colonies resulting from the culture were inoculated in96 well microtiter plates containing nutrient broth and ampicillin.These were grown, overnight, with shaking, at 37° C. DNA was purifiedvia standard alkaline lysis, with one modification. Specifically, at theend of the procedure, supernatant was passed through a multi-screenfilter prior to precipitation. Precipitated DNA was resuspended inwater, and used as described in the following example.

EXAMPLE 2

The purified DNA described in example 1 was sequenced, usingcommercially available reagents, and well known methods.

Specifically, commercially available products were used to carry out Taqdye deoxy terminator cycle sequencing reactions, using M13 reverse andforward matching primers, which flanked the inserts of the clones.Reaction products were analyzed on a commercially available geneticanalyzer.

There were a total of 289,963 shotgun genomic sequences. Open readingframes were obtained from these, and assembled using phred/phrapsoftware, as described by Ewing, et al., Genome Res 8(3):186-194 (1998)incorporated by reference. The assembly yielded 2,042 contigs. The“Glimmer” program of Delcher, et al., Nucl. Acids Res 27:4636-4641(1999), incorporated by reference, was applied to the contigs, andyielded 5826 putative open reading frames. Each of these ORFs containedat least 90 base pairs, and overlapped other ORFs by 30 base pairs, or10% of the ORF size, at most. Any ORFs with “N” or “X,” which indicatedeither poor quality, or repetitive regions were discarded. The “PSORT”program described by Nakai, et al., Proteins: Structure, Function andGenetics 11:95-110 (1991), incorporated by reference, was used topredict protein location within the bacterium. Any ORF, with a PSORTscore of 0.1 or more (outer membrane or periplasmic space localization),were considered to be potentially useful.

Protein coding genes were identified using known algorithms GeneMark andGlimmer, as described by Delcher, et al., Nucl. Acids Res. 27:4636-4641(1999), incorporated by reference, after which the “PSORT” program,described by Nakai, et al., Proteins: Structure, Function and Genetics11:95-110 (1991), incorporated by reference, was used to predict proteinlocalization within the bacterium. The sequences chosen for furtheranalysis were those which the program predicted to encode surfaceassociated proteins. Such proteins are ideal candidates for generationof antibodies.

Once the relevant nucleotide sequences were identified, protocols weredeveloped to amplify them for further work.

In each case, a pair of oligonucleotide primers were developed tohybridize specifically to the target sequence of interest. In general,primers from 18-28 nucleotides long were used. In each case, the forwardprimer was then modified to add the sequence “CACC” at the 5′ end. Thisfacilitates directional cloning in the vector used. As a generalprinciple, the primers were designed to hybridize from 10-25 amino acidsaway from the start codon, so as to avoid hydrophobic regions that arecharacteristic of signal peptide sequences. The primer sequences followMolecular Weight (KDa) Primers LpL53 53.8 LpL53F:CACCACCAATGTGTTTGGTATAGCG LpL53R: CAGCGTTTTGTGATAAAATTAAC OMPL55 53.8OMPL55F: CACCGATGCTTACTACGGACTGGATG OMPL55R: CAGGAGTGTGATCGAGCTTG OMPL1615.9 OMPL16F: CACCCCGTGTTCTTTTGGTTTAGAT OMPL16R: TTCCAACAAATCGAATCATCTOMPL31 32 OMPL31F: CACCAAGAAGGATTCCAACGATGATG OMPL31R:TCTCCTGCTTGACAGCCGAC OMPL15 15.2 OMPL15F: CACCATGCGTGCTGTCAGTAGAGAAACOMPL15R: GTCGACATTGGCAGAATTTACG OMPL20 21.2 OMPL20F:CACCTTTGCACAATCCAAAGAGAAATG OMPL20R: TCATTTCCGAACCGGATGAC LpL23 18.5LpL23F: ACCATGGGATCCGCTCTTTTGGTTGATCCAGAG LpL23R:GAATTCCTAACAACCAGGACCTTCACAT LpL40 39 LpL40F:ACCATGGGACTCGAGACGCCTCCTCCTAAAGATCC LpL40R:CTCCATGGTCATTTCAAAACTTCTACGGGGC LpL22 22.8 LpL22F:CACCCCTTCGAGGTTGGAAATCG LpL22R: AATCGATGGATCACGTTACG OMPL17 18.4OMPL17F: CACCAAACCTGGATATGGAATGGC OMPL17R: TACAGAGGTAGAAGCGTTAGAAGOMPL30 29.2 OMPL30F: CACCAATCGACTTTTCACTGAGTTTCTT OMPL30R:CGAAAGTATCAAGAAGAACCGTA OMPL27 27.5 OMPL27F: CACCCAGGAAACGGAAAACGCTAAOMPL27R: CTTATTGTTTGCCGTAGGTTTC OMPL21 21.9 OMPL21F:CACCATGGGCGCTTTTAATCGG OMPL21R: CGGAACTAGGGAACTTTTCAAC OMPL22 20.6OMPL22F: CACCATCATTCCTTCGGGAAGTGAC OMPL22R: CCATTCTCTGTTGTTTGATCCC MPL1715.4 MPL17F: CACCGAAAGTCCCGTAAGGTTCAAA MPL17R: TGCAGGAGTTCCCACATTTTAMPL21 31.9 MPL21F: CACCACGTCTCAAAGTTACGCTTCAG MPL21R:TTCTCACCATCCAGCTCGG OMPAL21 20.6 OMPAL21F: CACCGAGCCTTCAACGCAAGAGCAAOMPAL21R: AACGTAAGACGTTGAGTTGCCACA OMPL63 64.3 OMPL63F:CACCACGATGATTCAGCCTACTTGG OMPL63R: GAAGTAGAACCGGAAGATTTATTT OMPL14 18.6OMPL14F: CACCGGATGCAAACAAGATCCAGTAG OMPL14R: GAAACGCCACTAAGTTAGATCACMPL36 35.1 MPL36F: CACCACGTCTTGTGCGTCGGTAGAG MPL36R:CCAAGTATTCTATTTATACGTCCGAG MPL39 30.8 MPL39F:CACCTCAGTAACTACTGGTCAGTGTAATG MPL39R: CACGTGTTAGTTCTTTGGTTG MPL40 43.2MPL40F: CACCTTGTTTTTAAAAAAAAGGAAAGC MPL40R: AACTAAGGAACCGGAGTTGC MPL2118.6 MPL21F: CACCGACATGCTTCCTACTTATTCCC MPL21R: CGCTAAAAGTATCACAATGGTAA

Table 1—Oligonucleotide primer sequences employed to amplify the targetnucleic sequences by PCR methodology. Molecular weights are from therecombinant proteins obtained in E. coli expression systems.

The amplification was carried out by combining template DNA (1.0 ng),nucleotide triphosphates (0.4 mM), Pfx polymerase (0.5 units, taken froma stock solution of 1 unit/μl), and 0.2 μM primers, at a pH of 8.0. Thetotal reaction volume was 50 μl.

PCR was then carried out in a thermocycler, where the DNA denaturationstep was carried out for 3 minutes at 94° C., followed by primer-DNAtemplate annealing at 55° C., for 20 seconds, and nucleotidepolymerization for 4 minutes at 68° C. This cycle was repeated, 45times, except that in the repeats, denaturation was for 20 seconds.

Following amplification, the sequences were cloned into a commerciallyavailable vector, “pENTR.TOPO.” This vector includes a kanamycinresistance sequence useful in E. coli selection a pUC ORI, two “attL1”and “attL2” sites for site-specific recombination, and a TOPO cloningsite for directional cloning of blunt end PCR products. Specifically,this is the sequence “CCCTT”, which serves as a topoisomerase I bindingsite.

Cloning was carried out according to manufacturer's instructions, whichcall for 15 ng of the PCR product, and 5 ng of pENTR.TOPO vector, in 5mM Tris buffer, pH 7.4, at a total volume of 10 μl. The reactionproceeded for 10 minutes, at room temperature, i.e., about 25° C.

Positive clones were obtained via culture in kanamycin containing LuriaBroth.

Following the reaction, the PCR products were integrated into thepENTR.TOPO vector, and were transferred to specific expression vectorsvery easily, via reaction at the forementioned attR1 and attR2 sites, inthe presence of LR Clonase.

Expression was facilitated by use of an expression vector derived fromT7, the commercially available pDEST17 vector. This vector includes anOR1, a promoter sequence to facilitate transcription of insertedsequences, a ribosomal binding site, and an ATG start codon.

In the practice of the invention, 2μ of the cloning mixture describedsupra, 15 ng of pDEST17, & 4 μl of LR Clonase enzyme mix were combinedin Tris-EDTA (10mM Tris-HCl, 0.1 mM EDTA) buffer, at pH 8, for 60minutes, at room temperature. After 60 minutes, proteinase K (4 μg) wasadded for 30 minutes, at 37° C., to inactivate enzymes. Positive cloneswere isolated from Luria Broth agar plates, which contained 20 μg/ml ofampicillin. DNA sequences, when cloned, included a sequence encoding sixHis residues, at the N terminus of the protein. This well knowntechnique was used to facilitate the purification of protein via metalaffinity chromatography.

EXAMPLE 3

The expression vectors resulting from the preceding examples were usedto express the relevant proteins. E. coli strains BL21(DE3) or BL21SIwere used under inducing conditions, including 1 mM IPTG, or 300 mMNaCl, respectively, using standard methods. Proteins were then analyzedon 10-20% SDS-PAGE gels, under denaturing conditions. Each of FIGS. 1-22presents an SDS-PAGE pattern for the proteins.

In addition to the SDS-PAGE work, the proteins were purified, by usingNi²+ chelating sepharose. Either proteins were mixed with charged beads,or were applied onto columns in a balanced salt buffer (0.1M Tris/0.3 MNaCl, pH 8.0). Any impurities were washed away using the same buffer,with a low concentration of imidazole (20-60 mM). Proteins of interestwere then eluted, using a buffer containing imidazole at a concentrationof from 0.75 to 1.0M.

EXAMPLE 4

Once the proteins were purified, they were tested for their ability tobind to antibodies in the sera of infected subjects.

Proteins were purified on 10-15% SDS-PAGE, and then blot transferred tonitrocellulose membranes. Protein was visualized with Ponceau staining.

The proteins were then contacted with either pooled sera obtained frompatients who had been diagnosed with leptospiroses, or with ratsimmunized with leptospira interrogans serovar Copenhageni. In the caseof rats, dilutions varied from {fraction (1/500)} to {fraction(1/1000)}. For patients, dilutions ranged from {fraction (1/100)} to{fraction (1/1000)}. As controls, sera from healthy individuals andnon-immunized rats were used.

After contact with the sera, a second antibody, either anti-rat oranti-human IgG, labeled with peroxidase was added, followed by O-phenyldiamine benzidine and hydrogen peroxide.

The results, shown in FIGS. 1-22, inclusive, show that all of theproteins reacted with sera from both sources.

In the discussion of the proteins which follows, signal andtransmembrane regions were based upon the presence of hydrophobic aminoacids, including Ile, Tyr, Val, Phe, Leu, Met and Ala, and Nielsen, etal., Protein Engineering 12:3-9 (1999), incorporated by reference. Eachdescription is followed by reference to where the sequence appeared inthe provisional application.

SEQ ID NO: 1 sets forth a polynucleotide segment encoding an amino acidsequence as set forth in SEQ ID NO: 2 with a predicted molecular weightof 52.7 KDa. It is a new Leptospiral lipoprotein, LpL53, according tothe rules described in Haake, Microbiology 146:1491-1504 (2000),incorporated by reference. It has a hydrophobic region of a signalpeptide from amino acid 4 to 10 characterized by the presence of Tyr,Leu, Ile, Phe, Leu, Phe, Ile, the lipoprotein signal peptidase cleavagesite from amino acid 11-14, with Leu, Phe, Ser, Asn, and the cysteine tobe lipidated at position 15 of the polypeptide sequence. (3909 & 3910)

SEQ ID NO: 3 sets forth a polynucleotide segment encoding an amino acidsequence as set forth in SEQ ID NO: 4 with a predicted molecular weightof 55 KDa. It has a signal peptide cleavage site from amino acid 1 to28. (3531 & 3532) SEQ ID NO: 5 sets forth a polynucleotide segmentencoding an amino acid sequence as set forth in SEQ ID NO: 6 with apredicted molecular weight of 15.8 KDa. It has a signal peptide cleavagesite from amino acid 1 to 38. (3489 & 3490) SEQ ID NO: 7 sets forth apolynucleotide segment encoding an amino acid sequence as set forth inSEQ ID NO: 8 with a predicted molecular weight of 30.9 KDa. It has asignal peptide cleavage site from amino acid 1 to 19. (3871 & 3872) SEQID NO: 9 sets forth a polynucleotide segment encoding an amino acidsequence as set forth in SEQ ID NO: 10 with a predicted molecular weightof 15 KDa. No match with an y deposited protein in gene bank was foundwith this amino acid sequence. It has a signal peptide cleavage sitefrom amino acid 1 to 19 and a transmembrane segment from amino acid 7 to29, partially overlapping the signal peptide sequence. (3973 & 3974)

SEQ ID NO: 11 is a polynucleotide segment encoding an amino acidsequence as set forth in SEQ ID NO: 12 with a predicted molecular weightof 20.1 KDa. It has a signal peptide cleavage site from amino acid 1 to20. (3679 & 3680)

SEQ ID NO: 13 corresponds to a polynucleotide segment encoding an aminoacid sequence as set forth in SEQ ID NO: 14 with a predicted molecularweight of 23 KDa. This polypeptide sequence is a newly identifiedLeptospiral lipoprotein, according to Haake, supra. It has a signalpeptide hydrophobic region from amino acid 6 to 15 (Ile, Val, Tyr, Val,Ile, Tyr, Leu, Phe, Leu, Ile), characterized by a higher proportion ofhydrophobic amino acids, a lipoprotein signal peptides from amino acid16 to 19 (Ser, Leu, Tyr, Gly) and a cysteine to be lipidated at position20 of the polypeptide sequence. (81 & 82)

SEQ ID NO: 15 sets forth a polynucleotide segment encoding an amino acidsequence as set forth in SEQ ID NO: 16 with a predicted molecular weightof 40.6 KDa. This polypeptide sequence is a newly isolated Leptospirallipoprotein, according to the rules described in Haake, supra. It has asignal peptide hydrophobic region from amino acid 7 to 16 (Ile, Leu,Phe, Val, Leu, Thr, Gly, Phe, Ile, Phe), characterized by a higherproportion of hydrophobic amino acids, a lipoprotein signal peptidesfrom amino acid 1 to 20 (Phe, Val, Ser, Ala) and a cysteine to belipidated at position 21 of the polypeptide sequence. (43 & 44)

SEQ ID NO: 17 corresponds to a polynucleotide segment encoding an aminoacid sequence as set forth in SEQ ID NO: 18 with a predicted molecularweight of 22.6 KDa. This polypeptide sequence is a new Leptospirallipoprotein, according to Haake, supra. It has a signal peptidehydrophobic region from amino acid 8 to 18 (Ile, Asn, Ile, Leu, Phe,Phe, Phe, Leu, Val, Tyr, Phe), a lipoprotein signal peptidase from aminoacid 19 to 22 (Leu, Leu, Phe, Gly) that conforms to the rules describedin Haake and a cysteine to be lipidated at position 23 of thepolypeptide sequence. (125 & 126)

SEQ ID NO: 19 corresponds to a polynucleotide segment encoding an aminoacid sequence as set forth in SEQ ID NO: 20 with a predicted molecularweight of 17.3 KDa. It has a signal peptide cleavage site from aminoacid 1 to 20. (3991 & 3992)

SEQ ID NO: 21 sets forth a polynucleotide sequence encoding an aminoacid sequence as set forth in SEQ ID NO: 22 with a predicted molecularweight of 30.9 KDa. It has a signal peptide cleavage site from aminoacid 1 to 27. (3521 & 3522)

SEQ ID NO: 23 sets forth a polynucleotide sequence encoding an aminoacid sequence as set forth in SEQ ID NO: 24 with a predicted molecularweight of 27.1 KDa. It has a signal peptide cleavage site from aminoacid 1 to 20. It has some similarity to proteins belonging to cytochromec family. Examples are: di-haem cytochrome c peroxidase and cytochromec, class I found in bacteria, such as Bacillus subtilis. (3533 & 3534)

SEQ ID NO: 25 corresponds to a polynucleotide sequence encoding an aminoacid sequence as set forth in SEQ ID NO: 26 with a predicted molecularweight of 20.9 KDa. It has a signal peptide cleavage site from aminoacid 1 to 20. (3561 & 3562)

SEQ ID NO: 27 corresponds to a polynucleotide segment encoding an aminoacid sequence as set forth in SEQ ID NO: 28 with a predicted molecularweight of 21.2 KDa. It has a signal peptide cleavage site from aminoacid 1 to 34. It has some similarity to prokaryotic N-terminalmethylation site found in bacterial general secretion pathway protein Gand bacterial type II secretion system protein I/J. Examples of bacteriaare E. coli, Xanthomonas campestris and Pseudomonas aeruginosa. (3675 &3676)

SEQ ID NO: 29 corresponds to a polynucleotide sequence encoding an aminoacid sequence as set forth in SEQ ID NO: 30 with a predicted molecularweight of 16.6 KDa. It has a signal peptide cleavage site from aminoacid 1 to 36. (3819 & 3820)

SEQ ID NO: 31 corresponds to a polynucleotide sequence encoding an aminoacid sequence as set forth in SEQ ID NO: 32 with a predicted molecularweight of 20.5 KDa. It has a signal peptide cleavage site from aminoacid 1 to 22. (3829 & 3830)

SEQ ID NO: 33 is a polynucleotide sequence encoding an amino acidsequence as set forth in SEQ ID NO: 34 with a predicted molecular weightof 20.9 KDa. It has a signal peptide cleavage site from amino acid 1 to22. This polypeptide has some similarity to proteins having an outermembrane domain (from amino acid 77 to 182) that belong to the OmpAfamily. Most of these bacterial outer membrane proteins in this groupare porin-like, integral membrane proteins, but some are smallpeptidoglycan-associated lipoprotein (such as pal). Escherichia coli isan example of a bacterium expressing a protein and having this domain.(3793 & 3794)

SEQ ID NO: 35 is a polynucleotide sequence encoding an amino acidsequence as set forth in SEQ.ID NO: 36 with a predicted molecular weightof 63.5 KDa. It has a signal peptide cleavage site from amino acid 1 to29. This polypeptide has some similarity to outer membrane effluxprotein identified in other bacteria. Examples include the E. coli TolCouter membrane protein; the Rhizobium nodulation protein; and thePseudomonas FusA protein. (4007 & 4008)

SEQ ID NO: 37 is a polynucleotide sequence encoding an amino acidsequence as set forth in SEQ ID NO: 38 with a predicted molecular weightof 14 KDa. It has a signal peptide cleavage site from amino acid 1 to23. (3883 & 3884)

SEQ ID NO: 39 is a polynucleotide sequence encoding an amino acidsequence as set forth in SEQ ID NO: 40 with a predicted molecular weightof 35.6 KDa. It has a signal peptide cleavage site from amino acid 1 to36. This polypeptide sequence has some similarity to bacteriallipoproteins family identified in bacteria, such as, Escherichia coli.(2019 & 2020)

SEQ ID NO: 41 is a polynucleotide sequence encoding an amino acidsequence as set forth in SEQ ID NO: 42 with a predicted molecular weightof 39.8 KDa. This protein has a transmembrane region from amino acid 68to 90. This sequence has similarity to DshA protein of leptospira withunknown function. (1031 & 1032)

SEQ ID NO: 43 is a polynucleotide sequence encoding an amino acidsequence as set forth in SEQ ID NO: 44 with a predicted molecular weightof 40 KDa. It is a cytoplasmic membrane protein, with one transmembranesegment from amino acid 63 to 79. This polypeptide sequence has somehomology to the MoxR protein identified in Borrelia burgdorferi. Theprotein family (St. Louis Pfam Web site) (http://pfam.wustl.edu/)predicted two domains in this protein: (i) ATPase family domainassociated with various cellular activities (AAA), such aschaperone-like functions that assist in the assembly, operation, ordisassembly of protein complexes. (2517 & 2518)

SEQ ID NO: 45 corresponds to a polynucleotide segment encoding an aminoacid sequence as set forth in SEQ ID NO: 46 with a predicted molecularweight of 21 KDa. This protein has a signal peptide cleavage site fromamino acid 1 to 40 and a transmembrane domain from amino acid 20 to 40.The polypeptide sequence has some similarity to bacterial signalpeptidases identified in bacteria, such as, Sinorhizobium meliloti andBacillus subtilis. This is a Leptospiral membrane protein MPL21. (1991 &1992)

The foregoing disclosure set forth various aspects of the invention,including the isolated, leptospira surface proteins, the amino acidsequences of which are set forth as an attachment hereto, and isolatednucleic acid molecules which encode these proteins such as those setforth herein. It will be understood that once an amino acid sequence isknown, various degenerate nucleotide sequences can be provided whichencode that sequence. All of those are encompassed by this invention.

“Surface protein” as used herein, refers to proteins which areassociated and/or exposed to the surface of the organism.

The proteins of the invention may be used, alone or in combination witheach other, as or as components of vaccines. Further, they can be usedas immunogens, so as to generate an antibody response. The antibodiesthus generated can be used, e.g., as diagnostic tools to determineleptospira infection or presence, as well as components of vaccines usedto generate passive immunity. Proteins and antibodies may beadministered “neat” or “compounded” with other standard materials usedin preparing vaccines, such as carriers, adjuvants, and other materials.Intravenous formulations are one embodiment and, it will be understoodthat other formulations of vaccines are possible, including intradermal,subcutaneous, oral, such as sublingual forms, and others. The vaccinesmay be in liquid or “dry” form, such as in lyophilized form. This typeof vaccine is especially suitable when it must be carried “in thefield,” and used at some point in time later than when carried.

The nucleic acid molecules of the invention, as will be understood bythe skilled artisan, can be used to produce the proteins describedsupra, via any of the recombinant methodologies well known to theskilled artisan. They can be placed in, e.g., expression vectors, undercontrol of a promoter or other regulatory element, or they can be used“as is” to transfect or to transform cells. Eukaryotic cells, such asyeast cells, CHO cells, fibroblasts, insect cells, etc., are among theeukaryotes which can be transformed or transfected. Prokaryotes, such asE. coli or other bacteria can also be used. The choice of host cell willdepend upon many factors, including whether or not glycosylation isdesired, and to what end the transformant or transfectant will be used.One way these recombinant cells can be used is as in the form of a cellbased vaccine, such as a whole cell vaccine. As the recombinant proteinsof the invention are all surface proteins, it is to be expected thatthey will be expressed on the surface of host cells. If the cells arethen processed so as to become non-proliferative, the cells present anideal vaccine, especially if the host cell is one that is not normallythe target of immune surveillance in the host.

Another aspect of the invention is the use of membrane preparations, orcellular “ghosts” of transformants or tranfectants. Such approaches havebeen used with other bacterial species, so preparation of these is wellknown. Transformants or transfectants which express the surface proteinsof the invention can be used to prepare these materials in a fashiontaught by the art.

The proteins have been discussed as vaccines, supra. It is to beunderstood that the vaccines of the invention can be formulated for anysubject. Human vaccines are, of course, included, but so are vaccinesfor livestock animals, such as sheep, bovine animals, goats, pigs and soforth, and domesticated animals such as pets, confined zoo animals,etcetera. The vaccines may be used prophylactically, e.g., byadministering them prior to possible exposure to Leptospira, and mayalso be used post exposure, in order to treat a pre-existing infection.

As will be recognized by the skilled artisan, the use of the proteins ofthe invention, or portions thereof, constitutes vaccination, and theprotein or portion of the protein constitutes the vaccine. Uponadministration to the individual or subject, in any of the waysdescribed supra, an immune response results which protects theindividual or subject when confronted with the pathogen. Such anapproach, i.e., the immunization with one or more proteins or portionsof proteins, can be used therapeutically or prophylactically. Passiveimmunization, as described supra, can also be used. In such a case,antibodies are developed against the proteins or protein portions, andvia passive transfer serve a role in, e.g., prophylaxis.

It is to be understood that, while immunization with the proteins orportions of proteins can serve to stimulate an antibody response,cellular immune responses are also a part of the response of the subjectto the immunization. It is well within the skill of the artisan todetermine which proteins or portions of proteins function as antibody orcellular immune vaccine agents and that artisan can then formulate,e.g., “cocktails” of appropriate mixes of proteins which have thedesired, immune effect.

In addition to the use of the proteins as vaccines, it is to beunderstood that the nucleic acid molecules described herein can also beused as vaccines. Via targeted delivery of nucleic acid molecules, onecan assure an “in vivo” supply of the desired protein or protein portionmolecules at a site of relevance. The artisan of delivery systems, suchas liposomes, adenoviruses, retroviruses, and other formats for thedelivery of DNA as immunoprophylactic or a therapeutic agents, and allare envisioned as methods for administering the vaccine. It should bekept in mind that the nucleic acid molecules of the invention includethose which encode the proteins of the invention, but differ innucleotide sequence due to codon degeneracy. Indeed, due to patterns ofcodon usage, which vary from organism to organism, it may be desirableto alter the sequence to maximize expression of the desired vaccine inthe treated subject.

To prepare a vaccine the purified polypeptide can be isolated,lyophilized and/or stabilized, as described supra. The protein may thenbe adjusted to an appropriate concentration, optionally combined with asuitable vaccine adjuvant, and packaged for use. Suitable adjuvantsinclude but are not limited to: surfactants and pluronic polyols;polyanions, e.g., pyran, dextran sulfate, poly IC; polyacrylics,carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, oilemulsions, vitamins, cytokines, hormones, aluminum, calcium salts, andmixtures thereof, bacterial and plant products, e.g., BacillusCalmette-Guerin (BCG), complete Freund's adjuvant, and threalose.Alternatively, the immunogenic protein may be incorporated intoliposomes for use in a vaccine formulation, may be fused to otherimmunogenic proteins, or may be conjugated to polysaccharides or otherpolymers. See Edelman, in New Generation Vaccines (Marshall Decker, N.Y.1997), incorporated by reference.

The weight of the immunogenic protein included in a given dosage ofvaccine can vary widely, e.g., from 5 ug-300 mg., and depends on: theage, weight, and physical condition of the animal or the human subjectconsidered for vaccination.

In addition, the nucleic acid molecules of the invention can be used asvaccines. The rational of this approach is that the DNA will produce theprotein following injection thus in turn inducing the desired immuneresponse.

For such vaccines, a pharmaceutical composition can include either amammalian recombinant expression vector, such as pTARGET, or a constructsuch as an expression vector, which includes a nucleic acid moleculeencoding the protein, operatively linked to transcriptioncontrol/terminator sequences, combined with a pharmaceuticallyacceptable carrier. These may include, but are not limited to, aqueousphysiologically balanced solutions, artificial lipid-containingsubstrates, natural lipid-containing substrates, oils, esters, andglycols. Pharmaceutically acceptable carriers can also include asuitable delivery vehicle, such as liposomes, micelles, and cells.Adjuvants for DNA based vaccines can be used, such as CpGoligonucleotides and cytonkines. The vaccines can also be delivered byattenuated bacteria, such as Salmonella.

Another feature of the invention is the use of the proteins, or portionsof the proteins of the invention, as well as nucleic acid molecules andportions of nucleic acid molecules, in the manufacture of kits usefulfor diagnosis of leptospira infection, either “in the field” or in thelaboratory. Such kits involve, e.g., the protein, protein portion,nucleic acid molecule, or nucleic acid molecule portion, in combinationwith, e.g., a solid phase, such as a bead, mutltiwell plate, etc., towhich the component is affixed. The kit can then be used by contacting asample of interest thereto, followed by a second component, which canalso be included in the kit, such as a labeled protein or proteinsportion, or labeled nucleic acid molecule or portion of a nucleic acidmolecule. When nucleic acid molecules are used in the diagnostic kits ofthe invention, it is desirable that each portion hybridize to a separateportion of a target nucleic acid molecule.

It may be desirable to screen the proteins and nucleic acid molecules ofthe invention in, e.g., and animal or cellular model prior toadministration to large animals or humans. Standard practice tests apotential vaccine in, e.g., a hamster, mouse, rat or other rodent modelto determine it efficacy and strength, and the molecules of thisinvention may be so tested as well. Testing of the DNA molecules in,e.g., microorganisms such as E. coli or other prokaryotes or eukaryoteorganisms can be carried out to deter mine which are, in fact, goodproducers, i.e., molecules which produce high-yields, and/or producetransformants which react well to culture. Other aspects of theinventions will be clear to the skilled artisan, and need not bereiterated here.

1-6. (canceled).
 7. An isolated Leptospiral protein consisting of theamino acid sequence SEQ ID NO:
 14. 8. Vaccine comprising the isolatedLeptospiral protein of claim 7, and a pharmaceutically acceptableadjuvant or carrier.
 9. An isolated antibody which specifically binds tothe isolated Leptospiral protein of claim
 7. 10. The isolated antibodyof claim 9, wherein said antibody is a monoclonal antibody. 11.Hybridoma cell line which produces the isolated antibody of claim 10.12-17. (canceled)